谷蠹RAPD反应体系与程序的优化

以谷蠹为研究对象,采用L16（45）正交组合实验和单因素梯度实验对MgCl2、dNTP、随机引物、Taq酶、模板DNA浓度和退火温度、循环次数等影响RAPD扩增的重要因素进行优化,以期建立最优的RAPD反应体系与程序。实验结果表明：各因素最适条件为：25μL PCR反应体系中,10×Buffer 2.5μL,MgCl22.5 mmol/L,dNTP0.4 mmol/L,随机引物960 pmol/L,Taq酶0.5 U,模板DNA 20ng;退火温度为37℃,循环次数为45次。在对谷蠹进行RAPD分析时,采用优化的反应条件,规范实验操作,使用同厂家同批次的试剂,实验结果就会具有很好的重复性和稳定性。

OPTIMIZATION OF THE SYSTEM AND PROCEDURE OF R.DOMINICA RAPD REACTION

Regarded R.dominica as the research object,several important factors included MgCl2,dNTP,random primer,Taq polymerase,template DNA and annealing temperature,amplification were optimized by L16（45）orthogonal design experiment and single factor experiment in order to establish optimal RAPD system and procedure.The results showed that a total volume of 25μL PCR reaction system conditioned 10×Buffer 2.5 μL,MgCl2 2.5 mmol/L,dNTP 0.4 mmol/L,random primer 0.9 μmol/L,Taq polymerase 0.5 U,template DNA 20ng,and annealing temperature was 37℃,amplification for 45 cycles.As the result,the RAPD can be repeated with optimized reaction condition,the same reagent and standard operation.